RESUMO
In cancers, apoptosis evasion through dysregulation of pro-apoptotic and anti-apoptotic intracellular signals is a recurring event. Accordingly, selective inhibition of specific proteins represents an exciting therapeutic opportunity. Myeloid cell leukemia 1 (MCL1) is an anti-apoptotic protein of the BCL-2 family, which is overexpressed in many cancers. Here, we demonstrate that MCL1 can be modified by the small ubiquitin-like modifier (SUMO) at K234 and K238 sites. The SUMOylation of MCL1 can improve its stability by inhibiting the MCL1 ubiquitin-proteasome pathway mediated by the Tripartite motif-containing 11 (TRIM11, a novel MCL1 ubiquitin E3 ligase that we identify in this study). Moreover, SUMOylation of MCL1 increases the proliferation of cancer cells by inhibiting apoptosis. These results suggest that the SUMOylation of MCL1 may play a significant role in the regulation of its function.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Animais , Apoptose , Células COS , Proliferação de Células , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma , SumoilaçãoRESUMO
Sphingolipid metabolism is increasingly recognised as a therapeutic target in cancer due to its regulation of cell proliferation and apoptosis. The sphingolipid rheostat is proposed to control cell fate through maintaining balance between pro-apoptotic and pro-survival sphingolipids. This balance is regulated by metabolising enzymes involved in sphingolipid production. One such enzyme, sphingosine kinase-2 (SPHK2), produces pro-survival sphingosine 1-phosphate (S1P) by phosphorylation of pro-apoptotic sphingosine. Elevated SPHK2 has been found in multiple cancer types and contributes to cell survival, chemotherapeutic resistance and apoptosis resistance. We have previously shown elevation of S1P in large granular lymphocyte (LGL) leukaemia serum and cells isolated from patients. Here, we examined SPHK2 expression in LGL leukaemia and found SPHK2 mRNA and protein upregulation in a majority of LGL leukaemia patient samples. Knockdown of SPHK2 with siRNA in LGL leukaemia cell lines decreased proliferation. Additionally, the use of ABC294640 or K145, both SPHK2-specific inhibitors, decreased viability of LGL leukaemia cell lines. ABC294640 selectively induced apoptosis in LGL cell lines and freshly isolated LGL leukaemia patient cells compared to normal controls. Mechanistically, SPHK2 inhibition downregulated pro-survival myeloid cell leukaemia-1 (Mcl-1) protein through proteasomal degradation. Targeting of SPHK2 therefore provides a novel therapeutic approach for the treatment of LGL leukaemia.
Assuntos
Leucemia Linfocítica Granular Grande/enzimologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Proteínas de Neoplasias/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Adamantano/análogos & derivados , Adamantano/farmacologia , Adulto , Idoso , Apoptose/efeitos dos fármacos , Indução Enzimática , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucócitos Mononucleares/enzimologia , Lisofosfolipídeos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas , Piridinas/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Esfingosina/análogos & derivados , Tiazolidinedionas/farmacologia , Regulação para CimaRESUMO
MiR-125b regulates the kinds of cells that undergo apoptosis physiologically and pathologically. However, whether miR-125b affects the apoptotic behavior of trophoblasts and the underlying molecular regulatory mechanisms remains unclear. This study investigated the effect of miR-125b on apoptosis of HTR-8/SVneo cells in vitro. Constructed wild-type reporter vector (Wt-3'UTR) or mutated type reporter vector (Mut-3'UTR) reporter plasmids were transiently transfected into 293â¯T cells along with miR-125b mimics or a negative control. The luciferase reporter assay was used to validate whether the predicted MCL1 gene is a direct target of miR-125b. The HTR8/SVneo cells were transfected with miR-125 mimics, inhibitors, or a scramble control. Real-time polymerase chain reaction and western blotting were used to analyze mRNA and protein expression of the target gene MCL1. Flow cytometry was used to determine the effects on apoptosis. The luciferase activity assay validated the ability of miR-125b to specifically attenuate MCL1 transcription in the 293â¯T cell line, suggesting that MCL1 is a direct target of miR-125b. After transfection by miR-125b, relative expression and translation of the target gene MCL1 mRNA were repressed in HTR-8/SVneo cells. Trophoblast cells were induced to undergo apoptosis by overexpressing miR-125b in the HTR-8/SVneo cell line. In conclusion, MiR-125b may induced apoptosis of HTR8/SVneo cells by targeting MCL1. Our findings suggest that miR-125b may play a pivotal role in the pathophysiology of placentation.
Assuntos
Apoptose , MicroRNAs/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Trofoblastos/fisiologia , Linhagem Celular , HumanosRESUMO
Cisplatin alone or in combination with 5FU (5-fluorouracil) and docetaxel (TPF) are common regimen chemotherapeutics for treatment of advanced oral squamous cell carcinoma (OSCC). Despite the initial positive response, several patients experience relapse due to chemoresistance. The potential role of Bcl-2 antiapoptotic members in acquired chemoresistance is yet to be explored. To address this, we designed two different relevant OSCC chemoresistant models: (i) acquired chemoresistant cells, where OSCC lines were treated with conventional chemotherapy for a prolonged period to develop chemoresistance, and (ii) chemoresistant patient-derived cells, where primary cells were established from tumor of neoadjuvant-treated OSCC patients who do not respond to TPF. Among all Bcl-2 antiapoptotic members, Mcl-1 expression (but not Bcl-2 or Bcl-xL) was found to be upregulated in both chemoresistant OSCC lines and chemoresistant tumors when compared with their respective sensitive counterparts. Irrespective of all three chemotherapy drugs, Mcl-1 expression was elevated in OSCC cells that are resistant to either cisplatin or 5FU or docetaxel. In chemoresistant OSCC, Mcl-1 mRNA was upregulated by signal transducer and activator of transcription 3 (STAT3) activation, and the protein was stabilized by AKT-mediated glycogen synthase kinase 3 beta (GSK3ß) inactivation. Genetic (siRNA) or pharmacological (Triptolide, a transcriptional repressor of Mcl-1) inhibition of Mcl-1 induces drug-mediated cell death in chemoresistant OSCC. In patient-derived xenograft model of advanced stage and chemoresistant OSCC tumor, Triptolide restores cisplatin-mediated cell death and facilitates significant reduction of tumor burdens. Overall, our data suggest Mcl-1 dependency of chemoresistant OSCC. A combination regimen of Mcl-1 inhibitor with conventional chemotherapy deserves further clinical investigation in advanced OSCC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Glicogênio Sintase Quinase 3 beta/fisiologia , Neoplasias Bucais/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Fator de Transcrição STAT3/fisiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Diterpenos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Compostos de Epóxi/farmacologia , Fluoruracila/uso terapêutico , Humanos , Masculino , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Taxoides/uso terapêuticoRESUMO
BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses.
Assuntos
Flavivirus/patogenicidade , Proteína bcl-X/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Vírus da Dengue/patogenicidade , Vírus da Dengue/fisiologia , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Flavivirus/fisiologia , Infecções por Flavivirus/genética , Infecções por Flavivirus/patologia , Infecções por Flavivirus/fisiopatologia , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Imunidade Inata , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Células U937 , Células Vero , Replicação Viral/fisiologia , Zika virus/patogenicidade , Zika virus/fisiologia , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genéticaRESUMO
BACKGROUND: The development of approaches that increase therapeutic effects of anti-cancer drugs is one of the most important tasks of oncology. Caloric restriction in vivo or serum deprivation (SD) in vitro has been shown to be an effective tool for sensitizing cancer cells to chemotherapeutic drugs. However, the detailed mechanisms underlying the enhancement of apoptosis in cancer cells by SD remain to be elucidated. METHODS: Flow cytometry, caspase activity assay and western blotting were used for cell death rate evaluation. Western blotting, gel-filtration, siRNA approach and qRT-PCR were used to elucidate the mechanism underlying cell death potentiation upon SD. RESULTS: We demonstrated that SD sensitizes cancer cells to treatment with chemotherapeutic agent cisplatin. This effect is independent on activation of caspases-2 and -8, apical caspases triggering apoptosis in response to genotoxic stress. SD potentiates cell death via downregulation of the anti-apoptotic protein Mcl-1. In fact, SD reduces the Mcl-1 mRNA level, which consequently decreases the Mcl-1 protein level and renders cells more susceptible to apoptosis induction via the formation of apoptosome. CONCLUSIONS: Mcl-1 protein is an important regulator of sensitivity of cancer cells to apoptotic stimuli upon SD. GENERAL SIGNIFICANCE: This study identifies Mcl-1 as a new target for the sensitization of human cancer cells to cell death by SD, which is of great significance for the development of efficient anti-cancer therapies.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteínas de Neoplasias/biossíntese , Apoptose/fisiologia , Apoptossomas/fisiologia , Caspase 2/fisiologia , Caspase 8/fisiologia , Linhagem Celular Tumoral , Cisteína Endopeptidases/fisiologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Células HeLa , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose , Basófilos/metabolismo , Mastócitos/metabolismo , Compostos de Anilina/farmacologia , Animais , Basófilos/citologia , Basófilos/efeitos dos fármacos , Basófilos/enzimologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Caspase 3/metabolismo , Sobrevivência Celular , Humanos , Interleucina-3/antagonistas & inibidores , Interleucina-3/farmacologia , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidoresRESUMO
Fucoxanthin is a natural carotenoid that had never been previously demonstrated to have anti-tumor effect on human gastric adenocarcinoma SGC-7901 or BGC-823 cells. Here it was found to inhibit proliferation and induce apoptosis through JAK/STAT signal pathway in these cells; the mechanism by which this occurred was investigated. We find that fucoxanthin significantly increased the number of apoptotic cells by propidium iodide (PI) dye staining and flow cytometry. Fucoxanthin (50 or 75 µM) induced SGC-7901 cells cycle arrest at S phase, while BGC-823 cells arrest at G2/M phase. RT-PCR and western blot analysis revealed that the expressions of Mcl-1, STAT3 and p-STAT3 were obviously decreased by fucoxanthin in a dose-dependent manner. Synthetic siRNA targeting Mcl-1 was transfected into cells which had no effect on expressions of STAT3. After pretreatment with AG490 (50 µM) which led to blocking of the JAK/STAT signal pathway, the reductive expressions of Mcl-1, STAT3 and p-STAT3 caused by fucoxanthin were inhibited. This is the first analysis of effects on SGC-7901 and BGC-823 cells by fucoxanthin. Fucoxanthin can induce cell-cycle arrest and apoptosis in these cells. These effects involved downregulation of Mcl-1, STAT3 and p-STAT3. This work is significant for better understanding of mechanisms leading to human gastric adenocarcinoma formation and informing exploitation of anti-tumor marine drug, and for providing Mcl-1 and STAT3 as potential therapeutic targets for gastric adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Antineoplásicos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/fisiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Xantofilas/farmacologia , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Phaeophyceae , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Programmed apoptotic cell death is critical to maintain tissue homeostasis and cellular integrity in the lymphatic system. Accordingly, the evasion of apoptosis is a critical milestone for the transformation of lymphocytes on their way to becoming overt lymphomas. The anti-apoptotic BCL-2 family proteins are pivotal regulators of the mitochondrial apoptotic pathway and genetic aberrations in these genes are associated with lymphomagenesis and chemotherapeutic resistance. Pharmacological targeting of BCL-2 is highly effective in certain indolent B-cell lymphomas; however, recent evidence highlights a critical role for the BCL-2 family member MCL-1 in several lymphoma subtypes. MCL-1 is recurrently highly expressed in various kinds of cancer including non-Hodgkin's lymphoma of B- and T-cell origin. Moreover, both indolent and aggressive forms of lymphoma require MCL-1 for lymphomagenesis and for their continued survival. This review summarizes the role of MCL-1 in B- and T-cell lymphoma and discusses its potential as a therapeutic target.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Linfoma/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Humanos , Linfócitos/patologia , Linfoma/etiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologiaRESUMO
T lymphocyte non-Hodgkin's lymphoma (T-NHL) represents an aggressive and largely therapy-resistant subtype of lymphoid malignancies. As deregulated apoptosis is a frequent hallmark of lymphomagenesis, we analyzed gene expression profiles and protein levels of primary human T-NHL samples for various apoptotic regulators. We identified the apoptotic regulator MCL-1 as the only pro-survival BCL-2 family member to be highly expressed throughout all human T-NHL subtypes. Functional validation of pro-survival protein members of the BCL-2 family in two independent T-NHL mouse models identified that the partial loss of Mcl-1 significantly delayed T-NHL development in vivo. Moreover, the inducible reduction of MCL-1 protein levels in lymphoma-burdened mice severely impaired the continued survival of T-NHL cells, increased their susceptibility to chemotherapeutics and delayed lymphoma progression. Lymphoma viability remained unaffected by the genetic deletion or pharmacological inhibition of all alternative BCL-2 family members. Consistent with a therapeutic window for MCL-1 treatment within the context of the whole organism, we observed an only minimal toxicity after systemic heterozygous loss of Mcl-1 in vivo. We conclude that re-activation of mitochondrial apoptosis by blockade of MCL-1 represents a promising therapeutic strategy to treat T-cell lymphoma.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose , Linfoma de Células T/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/análise , Animais , Proteínas Reguladoras de Apoptose/análise , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Humanos , Linfoma de Células T/patologia , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Hypoxia is a common feature of solid tumors. Prolyl hydroxylase enzymes (PHD1-3) are molecular oxygen sensors that regulate hypoxia-inducible factor activity, but their functions in metastatic disease remain unclear. Here, we assessed the significance of PHD enzymes during the metastatic spread of colorectal cancer. PHD expression analysis in 124 colorectal cancer patients revealed that reduced tumoral expression of PHD3 correlated with increased frequency of distant metastases and poor outcome. Tumorigenicity and metastatic potential of colorectal tumor cells over and underexpressing PHD3 were investigated in orthotopic and heterotopic tumor models. PHD3 overexpression in a syngeneic tumor model resulted in fewer liver metastases, whereas PHD3 knockdown induced tumor spread. The migration of PHD3-overexpressing tumor cells was also attenuated in vitro Conversely, migratory potential and colony formation were enhanced in PHD3-deficient cells, and this phenotype was associated with enhanced mitochondrial ATP production. Furthermore, the effects of PHD3 deficiency were accompanied by increased mitochondrial expression of the BCL-2 family member, member myeloid cell leukemia sequence 1 (MCL-1), and could be reversed by simultaneous inhibition of MCL-1. MCL-1 protein expression was likewise enhanced in human colorectal tumors expressing low levels of PHD3. Therefore, we demonstrate that downregulation of PHD3 augments metastatic spread in human colorectal cancer and identify MCL-1 as a novel downstream effector of oxygen sensing. Importantly, these findings offer new insight into the possible, context-specific deleterious effects of pharmacologic PHD inhibition. Cancer Res; 76(8); 2219-30. ©2016 AACR.
Assuntos
Trifosfato de Adenosina/biossíntese , Neoplasias Colorretais/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Metástase Neoplásica , Prolil Hidroxilases/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
Chronic hepatitis, including viral hepatitis and steatihepatitis, is a well-known high-risk condition for hepatocellular carcinoma. We previously reported that continuous hepatocyte apoptosis drives liver tumors in hepatocyte-specific Bcl-xL or Mcl-1 knockout mice. In this study, we further examine the underlying cellular mechanisms of generating tumors in apoptosis-prone liver. In cultured hepatocytes, the administration of ABT-737, a Bcl-xL/-2/-w inhibitor, led to production of reactive oxygen species (ROS) as well as activation of caspases. Mitochondria isolated from murine liver, upon administration of truncated-Bid, a proapoptotic Bcl-2 family protein, released cytochrome c and produced ROS, which was dependent on mitochondrial respiration. Hepatic apoptosis, regeneration, accumulation of oxidative damages, and tumorigenesis observed in hepatocyte-specific Mcl-1 knockout mice were substantially attenuated by further deficiency of Bax or Bid, suggesting that a balance of mitochondrial Bcl-2 family proteins governs generation of oxidative stress and other pathologies. Whole-exome sequencing clarified that C>A/G>T transversion, which is often caused by oxidative DNA damage in proliferating cells, was a frequently observed mutation pattern in liver tumors of Mcl-1 knockout mice. The administration of antioxidant L-N-acetylcysteine did not affect apoptosis, compensatory regeneration, or fibrotic responses but significantly reduced oxidative DNA damage and incidence and multiplicity of live tumors in Mcl-1 knockout mice. In conclusion, activation of the mitochondrial apoptotic pathway in hepatocytes accumulates intracellular oxidative damages, leading to liver tumorigenesis, independently of liver regeneration or fibrosis. This study supports a concept that antioxidant therapy may be useful for suppressing liver carcinogenesis in patients with chronic liver disease.
Assuntos
Carcinogênese/patologia , Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Mitocôndrias/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Carcinogênese/metabolismo , Caspases/metabolismo , Proliferação de Células , Células Cultivadas , Hepatócitos/metabolismo , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2/fisiologiaRESUMO
HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound's ability to down-regulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells.
Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Naftoquinonas/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Transdução de Sinais/fisiologia , Survivina , Células U937RESUMO
A majority of ovarian follicles are lost to natural death, but the disruption of factors involved in maintenance of the oocyte pool results in a further untimely follicular depletion known as premature ovarian failure. The anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia-1 (MCL-1) has a pro-survival role in various cell types; however, its contribution to oocyte survival is unconfirmed. We present a phenotypic characterization of oocytes deficient in Mcl-1, and establish its role in maintenance of the primordial follicle (PMF) pool, growing oocyte survival and oocyte quality. Mcl-1 depletion resulted in the premature exhaustion of the ovarian reserve, characterized by early PMF loss because of activation of apoptosis. The increasingly diminished surviving cohort of growing oocytes displayed elevated markers of autophagy and mitochondrial dysfunction. Mcl-1-deficient ovulated oocytes demonstrated an increased susceptibility to cellular fragmentation with activation of the apoptotic cascade. Concomitant deletion of the pro-apoptotic Bcl-2 member Bcl-2-associated X protein (Bax) rescued the PMF phenotype and ovulated oocyte death, but did not prevent the mitochondrial dysfunction associated with Mcl-1 deficiency and could not rescue long-term breeding performance. We thus recognize MCL-1 as the essential survival factor required for conservation of the postnatal PMF pool, growing follicle survival and effective oocyte mitochondrial function.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Reserva Ovariana/fisiologia , Animais , Apoptose/fisiologia , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Oócitos/fisiologiaRESUMO
Apoptosis, cell death executed by caspases, is essential to normal breast development and homeostasis. Pro-apoptotic and anti-apoptotic signals are tightly regulated in normal breast epithelial cells. Dysregulation of this balance is required for breast tumorigenesis and increases acquired resistance to treatments, including molecularly targeted therapies, radiation and chemotherapies. The pro-apoptotic or anti-apoptotic Bcl-2 family members interact with each other to maintain mitochondrial integrity and regulate cellular commitment to apoptosis. Among the anti-apoptotic Bcl-2 family members, Mcl-1 is uniquely regulated by numerous oncogenic signaling pathways. This review will focus on the role of Bcl-2 family proteins in normal breast development, breast tumorigenesis and acquired resistance to breast cancer treatment strategies, while highlighting Mcl-1 as a promising target to improve breast cancer tumor cell killing.
Assuntos
Neoplasias da Mama/patologia , Mama/crescimento & desenvolvimento , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Apoptose/fisiologia , Mama/citologia , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Apoptosis plays a role in normal lymphopoiesis and lymphoid malignancies. Pro-survival MCL-1 is essential for survival of T-cell progenitors, BCL-XL for immature thymocytes, and BCL-2 for mature T cells. Conversely, little is known about the regulators that are required for the survival of T-cell lymphomas. We used constitutive and conditionally gene-targeted mice to investigate which pro-survival BCL-2 family member is required for the sustained survival of thymic lymphomas initiated by loss of p53. Constitutive loss of a single Mcl-1 allele delayed tumor onset. In contrast, lymphomas emerging in p53(-/-) mice in which Mcl-1 could be conditionally deleted had been selected for retention of MCL-1 expression. In contrast, complete loss of BCL-XL had no impact on lymphoma development in p53(-/-) mice. These results demonstrate that thymic lymphomas elicited by loss of p53 must arise from cancer-initiating cells that require MCL-1 for their survival. Acute deletion of both Mcl-1 alleles abrogated the expansion of p53(-/-) lymphomas in mice, whereas inducible loss of BCL-XL had little impact. This reveals that MCL-1 is essential for the sustained survival of these malignant cells and suggests that targeting MCL-1 may be an attractive strategy for the treatment of T-cell lymphoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes p53 , Linfoma de Células T/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Neoplasias do Timo/metabolismo , Proteína bcl-X/fisiologia , Alelos , Animais , Apoptose , Sobrevivência Celular , Transplante de Células , Deleção de Genes , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Células-Tronco/citologia , Proteína bcl-X/genéticaRESUMO
The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Proteína X Associada a bcl-2/fisiologia , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína 11 Semelhante a Bcl-2 , Benzamidas/química , Benzamidas/farmacologia , Benzopiranos/química , Benzopiranos/farmacologia , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Células HeLa , Humanos , Indóis , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Mimetismo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Nitrilas/química , Nitrilas/farmacologia , Nitrofenóis/química , Nitrofenóis/farmacologia , Permeabilidade , Piperazinas/química , Piperazinas/farmacologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , Pirróis/química , Pirróis/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonas/química , Sulfonas/farmacologia , Lipossomas Unilamelares/química , Proteína X Associada a bcl-2/química , Proteína bcl-X/química , Proteína bcl-X/fisiologiaRESUMO
OBJECTIVE: Ovarian cancer is the leading cause of death from gynecologic cancer, reflecting its often late diagnosis and its chemoresistance. We identified a set of microRNAs whose expression is altered upon BAG3 knockdown. Our primary objective was to examine the relationships between BAG3, miR-29b and Mcl-1, an antiapoptotic Bcl-2 family protein, in ovarian cancer cells. METHODS: Ovarian cancer cells were cultured and their responsiveness to paclitaxel was tested. Microarray analysis was performed to identify microRNAs differentially expressed in ES2 BAG3 knockdown ovarian cancer cells and their control cells. Primary ovarian cancer tissues were obtained from 56 patients operated on for ovarian cancer. The patients' clinical and pathological data were obtained from their medical records. RESULTS: BAG3 knockdown increased the chemosensitivity to paclitaxel of ES2 ovarian clear cell carcinoma cells to a greater degree than AMOC2 serous adenocarcinoma cells. qRT-PCR analysis showed that miR-29b expression was significantly upregulated in primary cancer tissue expressing low levels of BAG3, as compared to tissue expressing high levels. Moreover, levels of miR-29b correlated significantly with progression-free survival. Upregulation of miR-29b also reduced levels of Mcl-1 and sensitized ES2 cells to low-dose paclitaxel. CONCLUSIONS: BAG3 knockdown appears to downregulate expression of Mcl-1 through upregulation of miR-29b, thereby increasing the chemosensitivity of ovarian clear cell carcinoma cells. This suggests that BAG3 is a key determinant of the responsiveness of ovarian cancer cells, especially clear cell carcinoma, to paclitaxel and that BAG3 may be a useful therapeutic target for the treatment of ovarian cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adenocarcinoma de Células Claras/tratamento farmacológico , Antineoplásicos Fitogênicos/uso terapêutico , Proteínas Reguladoras de Apoptose/fisiologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/uso terapêutico , Regulação para Cima , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma de Células Claras/genética , Adulto , Idoso , Proteínas Reguladoras de Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias Ovarianas/genética , Adulto JovemRESUMO
Compared to the well-defined anti-apoptotic role of myeloid cell leukemia sequence 1 (MCL1), its antiproliferative function in tumorigenesis is less studied. We had recently reported that regulatory variants of MCL1 contribute to enhanced promoter activity but reduced risk of lung cancer. We hypothesized that MCL1 expression may manifest antiproliferative phenotype and its functional variations may have etiological relevance for breast cancer. We manipulated MCL1 expression in MCF-7 cells and MDA231 with overexpression and knockdown, analyzed the effects on cell viability and cell cycling phase, and characterized the correlation with expression profiles of key regulators of cell cycle. We further genotyped the -190 insertion polymorphism and the neighboring single nucleotide polymorphisms (SNPs) in 745 breast cancer patients and 537 controls and analyzed their association with cancer risk. We confirmed that heightened expression of MCL1 resulted in decreased proliferation ability of breast cancer cells. We further observed that MCL1 overexpression in breast cancer cells resulted in cell cycle progression arresting in S phase and concomitant enhanced expression of p27, which could be rescued by p27 knockdown with co-transfection of small interfering RNA (siRNA). Furthermore, we found a significant reduction in breast cancer risk [odds ratio (OR) = 0.74; 95 % confidence interval (CI) = 0.59-0.93] associated with -190 insertion genotype; the expression-enhancing regulatory haplotype (OR 0.79; 95 % CI 0.66-0.95) and diplotype (OR 0.71; 95 % CI 0.57-0.89) were consistently associated with decreased cancer susceptibility. The study demonstrates that the expression-enhancing regulatory variants of MCL1 are protective modifiers of breast cancer risk, and reduced cell proliferation and arrested cell cycle progression partly mediated by p27 might be the underlying mechanism.
Assuntos
Neoplasias da Mama/etiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Adulto , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/genéticaRESUMO
Temozolomide (TMZ) is an alkylating agent used for the treatment of glioblastoma multiforme (GBM), the main form of human brain tumours in adults. It has been reported that TMZ induced DNA lesions that subsequently trigger cell death but the actual mechanisms involved in the process are still unclear. We investigated the implication of major proteins of the Bcl-2 family in TMZ-induced cell death in GBM cell lines at concentrations closed to that reached in the brain during the treatments. We did not observe modulation of autophagy at these concentrations but we found an induction of apoptosis. Using RNA interference, we showed that TMZ induced apoptosis is dependent on the pro-apoptotic protein Bak but independent of the pro-apoptotic protein Bax. Apoptosis was not enhanced by ABT-737, an inhibitor of Bcl-2/Bcl-Xl/Bcl-W but not Mcl-1. The knock-down of Mcl-1 expression increased TMZ induced apoptosis. Our results identify a Mcl-1/Bak axis for TMZ induced apoptosis in GBM and thus unravel a target to overcome therapeutic resistance toward TMZ.